Agonist binding to the heptahelical luteinizing hormone/choriogonadotropin receptor (LH/CG R) activates the stimulatory guanine nucleotide binding protein (Gs) and downstream adenylyl cyclase (AC). The LH/CG R subsequently exhibits reduced hormone-dependent AC activity or desensitization in response to saturating agonist. Unlike desensitization of many G-protein coupled R's, the in vivo desensitization response of the LH/CG R in ovarian follicles to the preovulatory LH surge can be mimicked under cell-free conditions. Because LH/CG R desensitization in follicular membranes exhibits physiologically relevant kinetics without the addition of any exogenous proteins, the investigators can study the mechanisms involved in LH/CG R desensitization in an unperturbed membrane without complications associated with R internalization and recycling. Using this model the investigators have shown that desensitization of LH/CG R-stimulated AC activity requires GTP and is reversed by a GDP analog that prevents G protein activation, and that LH/CG R phosphorylation is not obligatory for LH/CG R desensitization. The investigators demonstrated that purified follicular membranes contain beta-arrestin and that neutralizing anti-arrestin antibodies specifically block development of desensitization. Addition of recombinant purified beta-arrestin mimicked hCG to promote LH/CG R desensitization. A synthetic peptide corresponding to the third intracellular (3i) loop of the LH/CG R specifically prevented desensitization and saturation of this peptide with exogenous beta-arrestin revived desensitization. These results lead the investigators to hypothesize that beta-arrestin participates in agonist-dependent desensitization of the LH/CG R in an apparent phosphorylation-independent manner by binding to the 3i loop of the LH/CG R to prevent activation of Gs.
The aims test this central hypothesis.
Aim 1 will test the hypothesis that beta-arrestin binds tightly and uniquely to the plasma membrane of ovarian follicles, perhaps in an FSH dependent manner.
Aim 2 will test the hypothesis that the binding of beta-arrestin to the active but unphosphorylated LH/CG R is necessary and sufficient to mediate LH/CG R desensitization.
Aim 3 will test the hypothesis that desensitization of the LH/CG R is associated with beta-arrestin-dependent LH/CG R oligomerization.
Aim 4 will test the hypothesis that the GTP-dependent step in LH/CG R desensitization consists of the release of beta-arrestin from a membrane docking site. Results from these studies will provide precise knowledge of the cellular mechanisms by which signaling from the LH/CG R is quenched to allow the reproductive cycle to advance and will likely permit development of reagents to halt the reproductive cycle or to reduce the activity of overactive R's.
|Hunzicker-Dunn, Mary; Barisas, George; Song, Jinming et al. (2003) Membrane organization of luteinizing hormone receptors differs between actively signaling and desensitized receptors. J Biol Chem 278:42744-9|
|Mukherjee, Sutapa; Gurevich, Vsevolod V; Preninger, Anita et al. (2002) Aspartic acid 564 in the third cytoplasmic loop of the luteinizing hormone/choriogonadotropin receptor is crucial for phosphorylation-independent interaction with arrestin2. J Biol Chem 277:17916-27|
|Hunzicker-Dunn, Mary; Birnbaumer, Lutz (2002) Cell-free membrane desensitization assay for G protein-coupled receptors. Methods Enzymol 343:394-404|
|Salvador, Lisa M; Maizels, Evelyn; Hales, Dale Buchanan et al. (2002) Acute signaling by the LH receptor is independent of protein kinase C activation. Endocrinology 143:2986-94|
|Mukherjee, S; Casanova, J E; Hunzicker-Dunn, M (2001) Desensitization of the luteinizing hormone/choriogonadotropin receptor in ovarian follicular membranes is inhibited by catalytically inactive ARNO(+). J Biol Chem 276:6524-8|
|Salvador, L M; Mukherjee, S; Kahn, R A et al. (2001) Activation of the luteinizing hormone/choriogonadotropin hormone receptor promotes ADP ribosylation factor 6 activation in porcine ovarian follicular membranes. J Biol Chem 276:33773-81|
|Mukherjee, S; Gurevich, V V; Jones, J C et al. (2000) The ADP ribosylation factor nucleotide exchange factor ARNO promotes beta-arrestin release necessary for luteinizing hormone/choriogonadotropin receptor desensitization. Proc Natl Acad Sci U S A 97:5901-6|